Isolation and Propagation of Human Corneal Stromal Keratocytes for Tissue Engineering and Cell Therapy

The human corneal stroma contains corneal stromal keratocytes (CSKs) that synthesize and deposit collagens and keratan sulfate proteoglycans into the stromal matrix to maintain the corneal structural integrity and transparency. In adult corneas, CSKs are quiescent and arrested in the G0 phase of the cell cycle. Following injury, some CSKs undergo apoptosis, whereas the surviving cells are activated to become stromal fibroblasts (SFs) and myofibroblasts (MyoFBs), as a natural mechanism of wound healing. The SFs and MyoFBs secrete abnormal extracellular matrix proteins, leading to corneal fibrosis and scar formation (corneal opacification). The issue is compounded by the fact that CSK transformation into SFs or MyoFBs is irreversible in vivo, which leads to chronic opacification. In this scenario, corneal transplantation is the only recourse. The application of cell therapy by replenishing CSKs, propagated in vitro, in the injured corneas has been demonstrated to be efficacious in resolving early-onset corneal opacification. However, expanding CSKs is challenging and has been the limiting factor for the application in corneal tissue engineering and cell therapy. The supplementation of serum in the culture medium promotes cell division but inevitably converts the CSKs into SFs. Similar to the in vivo conditions, the transformation is irreversible, even when the SF culture is switched to a serum-free medium. In the current article, we present a detailed protocol on the isolation and propagation of bona fide human CSKs and the morphological and genotypic differences from SFs.


Introduction
Corneal opacification is predominantly caused by scarring and haze development in the corneal stroma. The haze reduces and distorts the passage of light rays, leading to impaired vision [1,2]. Various factors including but not limited to traumatic injuries, infection, genetic, metabolic and developmental, and idiopathic causes can result in corneal opacification [3][4][5]. Affected individuals require corneal transplantation to restore vision [5]. However, the treatment approach is hindered by the global shortage of donor materials, long-term graft survival, immune response and need for surgical expertise, and high cost [6]. Our pre-clinical study has shown that stromal cell therapy via an intrastromal injection of human corneal stromal keratocytes (CSKs) enabled the repopulation of functional stromal cells in the cornea and reduction in stromal haze/opacities [7]. The possibility of cell propagation in vitro, therefore, can increase the amount of CSKs to achieve a "one donor to multiple recipients" strategy, and in turn, alleviate the issue of donor shortage. In addition, the simple cell delivery via intrastromal injection will be less demanding on surgical expertise and also allow faster visual rehabilitation.
Propagation of CSKs, however, is challenging because CSKs are typically quiescent in adult corneas [8]. To obtain a large number of CSKs for tissue engineering or cell therapy, the primary cells, after isolation from corneal stromal tissue, are often cultured in the presence of fetal bovine serum (FBS), typically in 5-10% concentration depending on the preferred growth stimulation [9][10][11][12][13]. However, this causes CSKs to become fibroblastic, similar to the in vivo corneal wound healing conditions, in that the surviving CSKs are activated to become stromal fibroblasts (SFs) due to the presence of serum proteins and growth factors that are released after the corneal epithelium and basement membrane are structurally compromised. The SFs lose CSK phenotype, which includes its quiescence, dendritic morphology, and expression of keratocan, lumican, and ALDH3A1, and also become proliferative and migratory that are accompanied by the upregulation of stress proteins and fibrosis-related and repair-type extracellular matrix (ECM) proteins (e.g., fibronectin, tenascin-C, and collagen type III), all of which are detrimental to corneal transparency when applied in vivo [10,11]. Although reversal of non-human SFs into CSKs in serum-depleted conditions in vitro resulted in partial recovery of CSK phenotypes, similar attempts to reverse human SFs into CSKs have largely been futile [14][15][16][17]. In many reversal instances, the CSK-specific dendritic morphology with extended cell processes was often lost, and the cells assumed a slender and fibroblastic appearance.
Studies have identified the potential of adult corneal stromal stem cells (CSSCs) in the peripheral cornea and limbus to adopt CSK phenotypes [18][19][20]. The stromal cells harvested near the limbal region, however, are typically heterogeneous. The lack of unique cellular markers makes the isolation of a homogenous and well-defined stem cell population that can differentiate into bona fide CSKs challenging [21,22]. We prefer expanding CSKs from the central corneal stroma. The stromal cell population in the central region is less heterogeneous and predominantly quiescent CSKs. The cells are less prone to fibroblastic transformation at low serum concentrations and easier to maintain while possessing similar wound healing and ECM remodeling properties to the CSSCs.
Our group has established a culture protocol to propagate human CSKs with a negligible transition to fibroblastic cells [11]. Our method starts with collagenase I digestion of corneal stromal tissue, followed by the culture of primary stromal cells in CSK basal medium with ERI supplement, which contains soluble human amniotic stromal Extract (AME) [23], Rho-associated coiled-coil-forming protein serine/threonine kinase inhibitor (Y-27632) [24], and Insulin-like growth factor 1 [25]. We referred the medium to as CSK complete medium. In the presence of low serum content (0.5% FBS) in the CSK complete medium, the "activated CSKs" can be propagated for 50-80 doublings. These cells have a unique genotype that features an expression of cell proliferation marker (PCNA), reduced expression of CSK-specific proteins (keratocan and lumican), and no expression of fibroblast-associated proteins (fibronectin and tenascin-C) [7,11,26]. They also feature suppressed fibroblast-induced collagen contractibility [11]. Once propagated, the "activated CSKs" culture is placed in serum-free CSK complete medium to drive the cells toward regaining quiescence, dendritic morphology, and unique CSK phenotypes. In the current protocol article, we have shown a step-by-step protocol to isolate, propagate, maintain, and cryopreserve the human CSKs for potential use in corneal tissue engineering and cell therapy.

Digestion Buffer Stock and Working Solutions
(1) Weigh 100 mg of collagenase I powder in a 15 mL centrifuge tube.
(2) Add 8 mL of 1× PBS and agitate the tube to dissolve the powder.
(3) Fill up the remaining 2 mL to make a final volume of 10 mL of 10 mg/mL collagenase type I. (4) Filter sterilize the collagenase I solution using Minisart ® RC syringe filter that is attached to a 10 mL syringe. Aliquot sterile collagenase I at 1 mL each into 1.5 mL microcentrifuge tubes and store the tubes of stock solutions at −20 • C * (* Note: Once thawed, collagenase should be stored at 4 • C. Repeat freeze-thawing should be avoided.) (5) Prepare 10 mL of digestion buffer working solution, containing 1 mg/mL collagenase I and 0.1% BSA by adding 1 mL of 10 mg/mL collagenase (prepared in Steps 1-5 above) and 10 mg of BSA to 9 mL of CSK basal medium which preparation protocol will be described in the following section *, **. (* Note: The digestion buffer working solution should be prepared fresh before cadaveric donor corneal tissue dissection.) (** Note: Adjust the volume of the digestion buffer accordingly based on the amount required for the corneal dissection procedure on the day. A central stromal tissue from one human cornea can typically be digested in 1 mL of digestion buffer working solution.)

CSK Complete Medium
(1) Add 9.6 mL of CSK basal medium in a 15 mL centrifuge tube.
(4) Add 50 of heat-inactivated FBS to obtain a final concentration of 0.5% *.
(5) Add 100 µL of 50 mM L-ascorbic 2-phosphate to obtain a final concentration of 0.5 mM *. (6) Add 5 µg/mL of AME and mix well (the AME preparation protocol will be elaborated in the Protocol section) *. (* Note: All supplements/growth factors should be added immediately before culture).

Overview of CSK Culture Procedure
The culture procedure should be initiated with the preparation of amnion extract, which takes 2 days at the most. Figure 1 displays the overview of the CSK isolation from donor corneal tissue to the conversion of activated CSKs into CSKs for cell-based therapies. Following amniotic protein extraction, the isolation of CSKs from cadaveric corneal tissue may proceed, which takes 1 day to complete. In the propagation phase, the isolated CSKs are cultured in CSK complete medium (containing 0.5% FBS) up to P6. Cryopreservation of activated CSKs can be carried out at any time between P3 and P6 (inclusive) when the cells reached~70% confluency. For cell therapy applications, the activated CSKs at P3-P6 are converted to CSKs by subjecting the cells to the stabilization phase in CSK complete medium (without 0.5% FBS) for 7-21 days. Overview of human corneal stromal keratocyte (CSK) cell culture procedure. In the propagation phase, the culture medium is supplemented with 0.5% fetal bovine serum (FBS) to support the proliferation of CSKs, which are otherwise quiescent. In the stabilization phase, the FBS is removed from the culture medium to allow the cells to regain bona fide CSK phenotypes.

Extraction of Proteins from Amniotic Membrane
The human amniotic membrane in the form of extract has been widely used to facilitate the propagation of various cell types, including corneal epithelial cells and CSKs [23,[27][28][29][30]. The effects of AME in the CSK culture system are multifactorial. The extract has been shown to be paramount in preventing fibroblastic transformation via the suppression of the TGF-β1/β2 pathway and is critical to eliminate fibroblast growth and maintain CSK characteristics in vitro [11,[30][31][32]. The amniotic membrane is also known to exert immunomodulatory effects, secrete proteins that support ECM and cytoskeleton remodelling, and modulate cell-matrix interactions that may be crucial in maintaining CSK phenotypes [11,[33][34][35][36]. AME can be derived either from fresh or cryopreserved human amniotic membrane; however, it is more likely for any lab to obtain a cryopreserved amniotic membrane. Amniotic membrane is typically cryopreserved in medium containing DMEM with 50% glycerol and penicillin/streptomycin [37]. We have shown that CSK cultures supplemented with extracts from either fresh or cryopreserved amnion samples resulted in similar cell morphology and viability, with only a marginal reduction in cell proliferation when AME from cryopreserved tissue was used [23]. We acknowledge that procurement of human amniotic membrane can be challenging in some countries. Sterilized γ-irradiated amniotic membrane is commercially available but its effect on the CSK culture has not been explored [38].
(Note: All procedures that require manipulation of amniotic membrane or its extract should be carried out in a tissue culture hood, with exceptions; the rotation and centrifugation steps. Since AME for cell culture purpose has to be prepared without the presence of protease inhibitors, all extraction steps must be kept in cool temperature or on ice to prevent intracellular enzyme activation, protein denaturation, and degradation.) (1) For fresh amniotic membrane, proceed to step 5.
(2) For cryopreserved amniotic membrane, thaw the vial containing the amniotic membrane at 4 • C overnight. (3) Prepare the tissue culture hood by UV sterilization for 15 min, followed by wiping off the work surface with 70% ethanol. (4) Turn on the germinator and heat-sterilize the forceps, scissors, and spatula. Ensure that forceps, scissors, and spatula have cooled down before handling them. (5) Wash the amnion 3-5 times in 1× PBS to remove traces of blood (for fresh amnion) or glycerol (for cryopreserved amnion). More washes have to be carried out if the amnion still has traces of blood. (6) Drain away PBS by squeezing the amnion with the forceps in a downward motion.
Repeat this a few times to remove as much PBS as possible for an easier grinding process later (PBS retention can cause ice formation in the amnion tissue when cooled with liquid nitrogen, leading to difficulty in grinding). (7) Place the amnion in a 60 mm cell culture dish and using mayo scissors, cut it intõ 1 cm 2 pieces. (8) Add liquid nitrogen to the mortar and pestle to cool it down. (9) Transfer 5-8 amnion pieces to the cooled mortar and add liquid nitrogen. (10) Grind the cooled amnion pieces using pestle into "powder" *, **, ***. (* Note: "Powder" is used as a term. The amnion will not be fully powderized. The grinding process only shears it into smaller fragments). (** Note: Due to the water retained by the membrane, the tissue will be hardened upon the addition of liquid nitrogen and challenging to grind. Allow the liquid nitrogen to evaporate before grinding to soften the amnion somewhat.) (*** Note: Before the membrane starts to become too soft, refill the mortar with liquid nitrogen. Do not let the membrane thaw fully.) (11) Transfer the amnion "powder" to a pre-weighed 50 mL centrifuge tube. Repeat Steps 8-11 until all amnion pieces have been processed. (12) Weigh the 50 mL centrifuge tube containing the amnion "powder" and mark down the weight of the "powder".

Culture of Human CSKs
Similar to in vivo, the CSKs isolated from human donor corneas inherently retain their quiescence nature in vitro. Hence, to produce sufficient number of CSKs for clinical translational purposes, 0.5% FBS has to be added to the culture medium (CSK complete media) to allow cell propagation ( Figure 3B,D). The propagation capacity of the cells, referred to as the activated CSKs, can be captured by the immunofluorescence staining of Ki-67. Before applying the cells for tissue engineering or cell therapy (i.e., intrastromal CSK injection), the activated CSKs have to be returned to quiescence ( Figure 3A,D). Hence, the cells are cultured in the CSK complete medium without FBS for 7-21 days to allow the cells to regain bona fide CSK morphology and phenotypes. We refer these cells to as the CSKs. Adding 5% FBS to the culture medium, significantly increase the cell proliferation but irreversibly transform the cells into SFs (Figure 3C,D). The proliferative capacity (indicated by Ki-67-positive cells/total number of cells × 100%) of the A-CSKs was 5.6 ± 6.1%. On day 14, following medium switching to serum-free conditions, the proliferative capacity of CSKs was 0%. The SFs had a significantly higher proliferation rate of 20.1 ± 7.2% compared to both the CSKs (p = 3.55 × 10 −8 ) and the activated CSKs (p = 3.78 × 10 −5 ). Group comparisons were statistically determined using one-way ANOVA and Tukey comparison tests. Scale bars = 100 µm.
(1) Filter the tissue digest from the preceding section (Isolation of CSKs from cadaveric donor cornea) using a 40 µm cell strainer into a 50 mL centrifuge tube to remove undissociated materials. Following that, wash the tube with 10 mL of 1× PBS once and pass it through the filter.

Passaging of Human CSKs and Preparation for Cell Therapy
The activated CSKs should be passaged once~70% confluency is reached. The morphology of cells tends to change into fibroblastic once the confluency exceeds 80%. This section of the protocol is also used to harvest CSKs in the preparation for tissue engineering or cell therapy applications.
(1) Aspirate the medium from the culture well.

Cryopreservation of Human CSKs
Cryopreservation of the activated CSKs should be performed when at least 4 wells of a 6-well plate have reached a confluency of~70% and when they are not planned for any immediate experiments. This is to minimize the passage number and thus, the propensity of cells to differentiate into fibroblasts at higher passage numbers.
(Note: Approximately 30-50% of frozen primary cells may lose viability after thawing. Therefore, it is not recommended to freeze down cells when the cell number is too low, such as less than 4-well of 6-well plate or less than 250,000 cells.) (1) Aspirate the culture medium from the well.

Cell Morphology
The differences in the cell morphology can be identified using phase-contrast microscopy. The human CSKs show thinner and distinctly more round-ish cell bodies ( Figure 4A,D) than the activated CSKs ( Figure 4B,E). The CSKs also feature longer cellular processes than the activated CSKs. The SFs, which are converted from the CSKs by the addition of 5% FBS in the CSK complete medium, show broader cell bodies with shorter cellular processes than both the CSKs and the activated CSKs ( Figure 4C,F). Further confirmation of phenotypic reversal of the cells can be achieved by staining with phalloidin. The human CSKs ( Figure 4G) assume a more stellate morphology with a concentrated F-actin organization near the end of the cellular processes compared to the activated CSKs ( Figure 4H). In stark contrast, the SFs are slender in shape and have a more bipolar morphology with larger cell bodies and an absence of cellular processes ( Figure 4I).

Protein and Gene Expression
The specific marker expression can be validated with immunofluorescence staining and real-time polymerase chain reaction (RT-PCR), which includes ALDH1A1, ALDH3A1, keratocan, and lumican (the antibodies, primers, and test procedures can be found in our previously published articles) [11,26]. The CSKs strongly express ALDH1A1 ( Figure 5A), ALDH3A1 ( Figure 5D), keratocan ( Figure 5G), and lumican ( Figure 5J), while the activated CSKs typically have an attenuated expression of these markers ( Figure 5B,E,H,K). This is attributed to the activated CSKs being cultured in the media containing 0.5% FBS for a prolonged period. When the cells from the same donor are cultured in CSK complete medium containing 5% FBS, the cells turn fibroblastic and lose the immunoreactivity to ALDH1A1 (Figure 5C Figure 6D). No significant difference in the level of ACTA2 expression is found in the CSKs, A-CSKs, and SFs ( Figure 6E). ACTA2 in all three cell types is significantly downregulated compared to MyoFBs (p = 3.16 × 10 −5 vs. CSKs; p = 2.63 × 10 −5 vs. A-CSKs; and p = 2.74 × 10 −5 vs. SFs) ( Figure 6E). For the analysis of differentially expressed genes, CSKs was used as the reference group for comparison, whereas GAPDH was used as the housekeeping gene. Group comparisons were statistically determined using one-way ANOVA and Tukey comparison tests.

Troubleshooting Guide
During the isolation of CSKs from donor corneal stroma, reagent preparations, and the culture of CSKs, some problems described in Table 1 may be encountered. The possible explanations and solutions are included in Table 1 to aid researchers in troubleshooting the problems. Table 1. Potential problems that may arise during CSK culture and their respective solutions.

Problem Encountered Explanations Solutions
Low amount of total protein in AME Amnion proteins are degraded • Ensure that the human amnion is placed in 4 • C or on ice before and during extraction.
• Store AME immediately after extraction procedure at −80 • C and thaw only when required.
Loss of proteins during processing • Ensure that amnion is processed as soon as possible from the time of collection/harvest.
The sample is diluted with a large volume of PBS after grinding • After filtering, wash the strainer with sufficient PBS to ensure most soluble proteins are collected.
• Reduce the volume of PBS added to the tube containing the AME "powder".
The sample is obtained from the anterior part of the amnion sac where the stroma is the thinnest

Microbial contamination
Aseptic techniques are not observed • Ensure that work surfaces are wiped with 70% ethanol, surgical instruments are heat-sterilized and all materials and reagents used are sterile.
• If contamination is minimal, remove the media and wash thrice with 1× PBS with antibiotic-antimycotic. Add media containing 2× antibiotic-antimycotic.
Donor corneal tissue is infected • If the cornea looks cloudy/hazy, do not process.

Conclusions
Besides describing the step-by-step procedures to isolate, culture, and propagate human CSKs, the current article also highlighted the complexity of CSK culture protocol, compared to the SF culture, which can be propagated in a simple medium that contains only DMEM/F-12 and 5% FBS [12,[39][40][41]. In addition, this article also highlighted the morphological and protein and gene expression differences between CSKs and SFs. The stark differences in the cellular morphology and phenotypes emphasized why the two cell types should not be used interchangeably in the literature and should not be therapeutically introduced in vivo before proper cell culture and propagation methods have been followed. Cell therapy with incorrect cell type could be detrimental to the patient's vision as we have shown in our rat experimental model [7]. With this protocol, we have greatly improved the efficiency of propagating CSKs and we can now increase the number of bona fide CSKs to achieve a "one donor to multiple recipients" strategy, and in turn, alleviate the issue of donor shortage. Our work suggests the plausibility of tissue engineering and cell-based therapy for treating corneal stromal disorders. Funding: This study was supported by the NMRC Clinician Scientist Award-Senior Category (MOH-000197-00).
Institutional Review Board Statement: Ethical review and approval were waived for this study, because the human donor corneas used to generate the data were procured from the Lions Eye Institute for Transplant and Research Inc. (Tampa, FL, USA).